This application is a 371 of PCT/EP97/07295 filed Dec. 23, 1997.
The principal objects of the present invention are:
novel cyclic organoselenium compounds;
the use of said novel compounds as antioxidant;
pharmaceutical compositions containing them;
a method of preparation of said novel compounds.
In aerobic organisms, during the metabolism of oxygen, very reactive entities are generated whose accumulation causes deleterious effects. These organisms possess a system of regulation, composed of enzymes and small is molecules which enable controlling the production of these reactive oxygen entities. Amongst the various components of this regulation system, often called Antioxidant Defense System, glutathione peroxidases play a central role in the prevention of  less than  less than oxidative stress greater than  greater than  and its deleterious consequences. These antioxidant and cytoprotecting enzymes enable degrading the endogenous or exogenous cytotoxic hydroperoxides.
These enzymes catalyse the reduction of hydrogen peroxide (reaction 1) or that of organic hydroperoxides (reaction 2) by reduced glutathione (GSH):
reaction 1:
H2O2+2GSHxe2x86x922H2O+GSSG
reaction 2:
ROOH+2GSHxe2x86x92ROH+H2O+GSSG
The active sites of these enzymes all contain one essential selenium atom in the form of a selenocysteine residue incorporated in the polypeptide chain.
The selenium is incorporated from selenite salts, or selenate salts, or L-seleno-methionine salts arising from food (see C. K. Chow and J. Jeng; in Selenium in Medicine and Biology, M. D. Spallholz and H. E. Ganther Eds.; (1986); Academic Press). In situations of selenium deficiency in the diet, the concentrations and activities of these glutathione peroxidases gradually decrease (see Y. X. Wang and J. Kiem.; Biological Trace Elements Res.; (1988); 15; 89 and see R. Reiter and A. Wendel; Biochem. Pharmacol.; (1983); 32; 3063-3067); this leads to an acute susceptibility to the oxidative stress (see D. B. Coursin and H. P. Cihla, Thorax, (1996), 51, 479-483). The provision of selenium in the diet is therefore a limiting factor in the biosynthesis of glutathione peroxidases.
The protecting role of glutathione peroxidases, in situations wherein the production of hydroperoxides rises, has been demonstrated following experiments of direct intracellular micro-injection of the enzyme of erythrocyte origin which have enabled demonstrating its cytoprotective effect upon the viability of fibroblasts or endothelial cells exposed to an oxidative stress (see C. to Michiels et al.; Experiment. Cell Res.; (1988); 179; 581-589). On the other hand, it has been shown that the survival of human fibroblasts is appreciably lowered when glutathione peroxidase is inhibited (see C. Michiels et al., J. Eur. Biochem., 1988, 177, 435-441).
Furthermore, the glutathione peroxidases are themselves particularly sensitive to an over-production of hydroperoxides and are rapidly inhibited under these conditions (see H. Ochi et al., Arch. of Biochem. Biophys., (1992), 294, 2, 407-411).
A certain number of pathologies such as certain ischemic cardiomyopathies for example (see J. Chaudixc3xa9re, in Biologie des lipides chez l""Homme, L. Doustes-Blazy and F. Mendy eds, Edition Mxc3xa9dicale Internationalexe2x80x94Paris, (1988), 137-154 and see D. Vitoux et al.; Ann. Biol. Clin.; (1996); 54; 5; 181-187) are associated with a lowering of glutathione peroxidase activity.
The demonstration of the essential role of the selenium at the active centre of these ubiquitous enzymes (see J. T. Rotruck, in Selenium in Biology and Medicine, Spallholz, J. E. Martin, J. L. Ganther H. I. eds., Avi Publishing Co, Wesport, (1981), 10-16) as well as the importance of the selenium in the regulation of oxidative damages generated during certain pathologies (see Cadenas, E. and Sies, H., Adv. Enz. Regul., (1985), 23, 217-237 and see Ursini, F., Bindoli, A., Chem. Phys. Lipids, (1987), 44, 255-276) has enabled the emergence of a novel class of organoselenium compounds as potential drugs.
Two types of compounds have been designed and prepared to this end.
On one side, modified macromolecules possessing a selenium atom introduced chemically such as selenosubtilisin (see Z. P. Wu and D. Hilvert, J. Am. Chem. Soc., (1990), 112, 5647-5648) or even a seleno-abzyme (see G. M. Luo et al., Biochem. Biophys. Res. Comm., 1994, 198, 3, 1240-1247). However, the use of proteins with a therapeutic aim is difficult to envisage for the following reasons:
their biostability is often insufficient;
an efficient method for ensuring their intra-cellular targeting does not exist;
they cannot be administered orally.
On the other side, synthetic molecules of low molecular weight have been synthetized, of which 2-phenyl-1,2-benzisoselenazolin-3-one (ebselen) (see H. Sies, Free Rad. Biol. Med., (1993), 14, 313-323) was the first compound described as having a glutathione peroxidase activity. Homologs of said ebselen, i.e. 2H-3,4-dihydro-1,2-benzoselenazin-3-ones, have also been described by Pierre V. Jacquemin et al. in Tetrahedron Letters, (1992), Vol. 33, No. 27, 3863-3866. In fact, several organoselenium derivatives have been described as glutathione peroxidase mimics, i. e. capable of reducing hydroperoxides in the presence of a biological thiol such as glutathione or lipoic acid (see I. A. Cotgreave et al., Biochem. Pharmacol., (1992), 43, 793-802 and C. M. Andersson et al., Free Rad. Biol. Med., (1994), 16, 17-28 and S. R. Wilson et al., J. Am. Chem. Soc., (1989), 111, 5936-5939 and V. Galet et al., J. Med. Chem., (1994), 37, 2903-2911). The patent application WO-A-95/27706 describes compounds of benzisoselenazoline and benzisoselenazine structure having a glutathione peroxidase activity inter alia.
These organoselenium compounds, which are mimics of glutathione peroxidase, invariably produce catalytic intermediates of the selenol and/or diselenide type.
Amongst these, 2-phenyl-1,2-benzisoselenazolin-3-one (ebselen) and some of its derivatives do not seem to have any major toxic effect (see A. Wendel et al.; Biochem. Pharmacol.; (1984); 33; 3241-3245 and S. D. Mercurio and G. F. Combs; Biochem. Pharmacol.; (1986); 35; 4505-4509). 2-Phenyl-1,2-benzisoselenazolin-3-one (ebselen) is however very little soluble in water, even in the presence of an excess of glutathione GSH, which limits its pharmacological applications.
The biochemical and pharmacological properties of the organoselenium compounds which have been synthesised and studied have been recently reviewed (see M. J. Pamham and E. Graf; Progress in Drug Res.; (1991); 36; 9-47 and M. J. Pamham, Exp. Opin. Invest. Drugs, (1996), 5, 7, 861-570).
One of the aims of the present invention is to design organoselenium compounds having a catalytic activity of the glutathione peroxidase type in the presence of physiological concentrations of glutathione GSH.
These compounds must be able to penetrate the target tissues or cells, be soluble in water at active concentrations and must not efficiently reduce oxygen into toxic by-products.
These aims are attained by virtue of the present invention which resides on the design of cyclic organoselenium compounds whose antioxidant and cytoprotecting activities have been demonstrated by the Applicant and which are given below.
The aim of the present invention is:
1) to solve the novel technical problem consisting of providing novel cyclic heteroaryl selenium compounds having a very good antioxidant and cytoprotecting activity, thus constituting valuable active principles of pharmaceutical compositions;
2) to solve the novel technical problem above according to a solution which includes a method of preparation of these novel compounds which is easy to carry out.
The technical problems set forth above are solved for the first time in a simultaneous manner by the present invention in a simple way; the method of preparation of said novel compounds being relatively easy to carry out and giving good yields.
According to its first aspect, the present invention thus relates to novel cyclic organoselenium compounds, having the general formula (I) below: 
in which:
R1=hydrogen; lower alkyl; optionally substituted aryl; optionally substituted lower aralkyl;
R2=hydrogen; lower alkyl; optionally substituted aryl; optionally substituted lower aralkyl;
A=CO; (CR3R4)m;
B=NR5; O; S;
Ar=optionally substituted phenyl or an optionally substituted radical of formula: 
xe2x80x83in which:
Z=O; S; NR5;
R3=hydrogen; lower alkyl; optionally substituted aryl; optionally substituted lower aralkyl
R4=hydrogen; lower alkyl; optionally substituted aryl; optionally substituted lower aralkyl;
R5=hydrogen; lower alkyl; optionally substituted aryl; optionally substituted lower aralkyl; optionally substituted heteroaryl ; optionally substituted lower heteroaralkyl; CO(lower alkyl); CO(aryl); SO2 (lower alkyl); SO2(aryl);
R6=hydrogen; lower alkyl; optionally substituted aryl; optionally substituted lower aralkyl; optionally substituted heteroaryl ; optionally substituted lower heteroaralkyl; trifluoromethyl; 
xe2x80x83m=0 or 1;
n=0 or 1;
X+ represents the cation of a pharmaceutically acceptable base; and their pharmaceutically acceptable salts of acids or bases with the proviso that:
when B=NR5 with R5 hydrogen, lower alkyl, optionally substituted lower aralkyl, CO(lower alkyl), and A=CO or (xe2x80x94CH2xe2x80x94)m, then Ar is different from an optionally substituted phenyl.
Said general formula (I) more particularly encompasses the four compounds, preparation of which is illustrated in the examples joined to this specification.
Said general formula (I) includes every stereoisomer, epimer and diastereoisomer, as a mixture or in isolated form.
It also includes, as indicated, the salts of pharmaceutically acceptable acids or bases of said compounds of formula (I).
Amongst the pharmaceutically acceptable acids, hydrochloric, hydrobromic, hydroiodic, sulphuric, tartaric, methanesulphonic, trifluoromethane-sulphonic acid, . . . can be cited in a non-limiting way.
Amongst the pharmaceutically acceptable bases, sodium hydroxide, potassium hydroxide, alkali metal or alkaline earth metal carbonates, or organic bases such as triethylamine or arginine, . . . can be cited in a non-limiting way.
Within the context of the present description and annexed claims:
the terms xe2x80x9clower alkyl and lower alkoxy (see below)xe2x80x9d are understood as meaning straight or branched alkyl and alkoxy groups having from 1 to 8 carbon atoms;
the term xe2x80x9carylxe2x80x9d is understood as meaning an aromatic group selected from phenyl and naphthyl groups;
the term xe2x80x9cheteroarylxe2x80x9d is understood as meaning a mono- or bicyclic aromatic group, each cycle, or ring, comprising five or six atoms and said cycle, or ring, or both cycles, or rings, including in its carbon skeleton from one to three heteroatoms selected from nitrogen, oxygen and sulphur;
the terms xe2x80x9clower aralkylxe2x80x9d and xe2x80x9clower heteroaralkylxe2x80x9d are understood as meaning, in view of the definitions above, phenyl(C1-C8)alkyl or naphthyl(C1-C8)alkyl and heteroar(C1-C8)alkyl respectively;
the terms xe2x80x9csubstitutedxe2x80x9d concerning the terms aryl, aralkyl, phenyl, radical (five-membered, including Z), heteroaryl, heteroaralkyl, as defined above, signifies that the groups in question are substituted on the aromatic part with one or more identical or different groups selected from the groups : (C1-C8)alkyl, trifluoromethyl, (C1-C8)alkoxy, hydroxy, nitro, amino, (C1-C8)alkylamino, di(C1-C8)alkylamino, sulphoxyl, sulphonyl, sulphonamide, sulpho(C1-C8)alkyl, carboxyl, carbalkoxyl, carbamide (it being possible for said (C1-C8)alkyl groups to be linear or branched) or substituted with one or more halogen atoms.
Finally, when R5 represents a hydrogen atom (B=xe2x80x94NH), the invention also includes the salts obtained with the pharmaceutically acceptable acids.
Said novel compounds have proved to be, as specified above, excellent antioxidant agents, the use of which is recommended by the Applicant in various fields. This use of said novel cyclic organoselenium compounds of the inventionxe2x80x94compounds having formula (I) as defined abovexe2x80x94as antioxidant agents, constitutes the second aspect of said invention.
Within the context of this second aspect, the use of said compounds of formula (I), is more particularly claimed as antioxidant agents:
intended to be added to preserving media of grafts for transplantation of organs of human or animal origin such as the heart, the liver, the kidney and the lungs; and
intended for (as active principle) the manufacture of pharmaceutical compositions with antioxidant activity, suitable especially:
for treatments of any physiopathological condition in which an over-production of cytotoxic hydroperoxides contributes to the functional impairments of cells or tissues; and more particularly including:
the treatment of inflammatory and/or ischemic cardio- and cerebro-vascular pathologies, such as the preventive and/or curative treatment of arterial restenoses following an angioplasty, the preventive and/or curative treatment of arterial stenoses following artery allografts, the treatment of intermittent claudication in patients affected with obstructive ischemia of the lower members, the treatment of cerebro-vascular accidents of ischemic origin;
the treatment of inflammatory and/or ischemic digestive pathologies, such as the treatment of acute inflammations of the bowel (Crohn""s disease, hemorrhagic rectocolitis);
the treatment of inflammatory and/or ischemic respiratory pathologies, such as the treatment of adult respiratory distress syndrome (ARDS) and infant respiratory distress syndrome (IRDS);
the treatment of inflammatory and/or ischemic ophthalmic pathologies, such as the treatment of glaucoma;
the treatment of cataracts;
the treatment of acute ophthalmic allergies
the treatment of impairments of the retina which are associated with a macular degeneration;
the treatment of viral infections causing an immuno-deficiency, such as the treatment of AIDS;
the treatment of post-radiotherapy fibroses.
Generally, the potential therapeutic applications of the compounds of the invention include the treatment of any physiopathological condition in which an over-production of cytotoxic hydroperoxides contributes to the functional impairments of cells or tissues. Such an over-production of hydroperoxides can be endogenous and secondary to the activation of the intra-cellular metabolic pathways such as, for example, those of the flavine or cytochrome P-450 oxygenases, those of the lipoxygenases, those of the monoamine oxidases. The over-production can also be due to the activation of the endothelial cells (xanthine oxidase, 15-lipoxygenase), or of blood platelets (cyclooxygenase and 12-lipoxygenase). It can also be due to the activation, by cytokines such as TNF-xcex1 for example, of inflammatory and/or immune cells such as neutrophils, macrophages or lymphocytes for example. It may also be due to an intoxication by a free-radical generating xenobiotic. Finally, it may be due to a voluntary irradiation such as practised during a radiotherapy, or an accidental irradiation.
More particularly, the second aspect of the present invention includes the use of compounds of the invention for the manufacture of pharmaceutical compositions intended for the treatment:
of inflammatory diseases of the bowel such as Crohn""s disease or hemorrhagic rectocolitis;
of adult respiratory distress syndrome and infant respiratory distress syndrome;
of cataracts;
of AIDS;
of post-radiotherapy fibroses.
From the second aspect of the present inventionxe2x80x94use of the novel compounds de formula (I) as antioxidant agentsxe2x80x94, such as described above, comes the third aspect which is dealt with now, namely the pharmaceutical compositions containing said compounds of formula (I) as active principle.
Thus, according to its third aspect, the present invention relates to pharmaceutical compositions, notably having an antioxidant activity, and comprising at least one cyclic organoselenium compound of the general formula (I), or one of its pharmaceutically acceptable salts of an acid or a base, as active ingredient, optionally incorporated in a pharmaceutically acceptable excipient, carrier or vehicle.
Said pharmaceutical compositions of the invention, according to an advantageous embodiment, contain said active ingredient in an amount between 0.1 and 5% by weight, advantageously between 0.1 and 1% by weight based on their total weight. According to another advantageous embodiment, said compositions are in the form of unit doses comprising from 1 to 500 mg of at least one cyclic organoselenium compound of the invention (optionally incorporated in a pharmaceutically acceptable excipient, carrier or vehicle).
The pharmaceutical compositions of the invention can be formulated for, or intended for, oral, rectal or topical administration, (the compounds of formula (I) may especially be formulated for ophthalmic applications in the form of an eye lotion) or even as intra-ventricular, intramuscular, subcutaneous or intravenous injections.
The pharmaceutically acceptable excipients, vehicles and carriers which can be included in their formulation are products which are well-known to the person skilled in the art and are not described in detail here.
The pharmaceutical compositions of the invention which contain the antioxidant agents disclosed by the present invention (compounds of formula (I)) are especially suitable for the treatment of any physiopathological condition in which an over-production of cytotoxic hydroperoxides contributes to the functional impairments of cells or tissues; it being possible for said over-production of hydroperoxides to be due to any one of the causes presented above in the present description, with reference to the second aspect of the invention (activation of the intra-cellular metabolic pathways, enzyme activation, macrophage or lymphocyte activation, intoxication by a free-radical generating xenobiotic, voluntary or accidental irradiation).
More specifically, said pharmaceutical compositions are suitable for the treatment of the pathologies listed above in the present description (with reference to the second aspect of the invention).
It is hereby specified that the antioxidant and therapeutical or pharmacological activities of the cyclic organoselenium compounds of the general formula (I) above have been demonstrated according to safe and reliable tests well-known to the person skilled in the art, which comprise:
a/ measuring the glutathione peroxidase activity;
b/ measuring the cytoprotective effect in human umbilical vein endothelial cells.
It is hereby incidentally mentioned that the preparation of pharmaceutical compositions incorporating an effective amount of at least one organoselenium compound of formula (I) according to the invention as well as the therapeutical treatments implying the use of such a compound make up an integral part of the present invention.
According to its last aspect, given below, the invention even relates to a method of preparation of said organoselenium compounds of formula (I). Said method comprises the following essential steps:
a/ preparing or using an orthohalo(hetero)arylacetonitrile derivative, optionally mono- or gem- disubstituted in the benzylic position; then, according to the series considered:
for the preparation of said compounds of formula (I) in which A=(CR3R4)m with m=0 (A does not exist) and n=0:
b1/ hydrolyzing said nitrile derivative into an amide derivative,
c1-1/ transforming this amide derivative into an amine derivative by a transposition reaction according to conventional methods,
d1-1/ allowing said amine derivative to react with a nucleophilic selenium derivative, optionally generated in situ, in the presence of a copper Cu(I) salt, in a polar organic solvent, to lead to the corresponding (hetero)arylisoselenazoline derivative,
e1-1/ optionally, N-alkylating or N-arylating or N-acylating or N-sulphonylating, according to conventional procedures, said (hetero)arylisoselen-azoline derivative;
for the preparation of said compounds of formula (I) in which A=CO and n=0:
b1/ hydrolyzing said nitrile derivative into an amide derivative,
c1-2/ allowing said amide derivative to react with a nucleophilic selenium derivative, optionally generated in situ, in the presence of a copper Cu(I) salt, in a polar organic solvent, to lead to the corresponding (hetero)arylisoselen-azone derivative,
d1-2/ optionally, N-alkylating or N-arylating or N-acylating or N-sulphonylating, said (hetero)arylisoselenazone derivative;
for the preparation of said compounds of formula (I) in which A=CH2 and n=0:
b2/ reducing said nitrile derivative into an amine derivative with the aid of borane for example in an ethereal solvent such as tetrahydrofuran for example,
c2/ allowing said amine derivative to react with a nucleophilic selenium derivative, optionally generated in situ, in the presence of a copper Cu(I) salt, in a polar organic solvent, to lead to the corresponding (hetero)arylisoselenazine derivative,
d2/ optionally, N-alkylating or N-arylating or N-acylating or N-sulphonylating, according to conventional procedures, said (hetero)aryliso-selenazine derivative;
for the preparation of said compounds of formula (I) in which A=(CR3R4) (xe2x89xa0CH2) and n=0:
b3/ carrying out a mono- or a bis-C-alkylation of said nitrile derivative according to conventional methods, with the aid of an organolithium derivative for example, in an ethereal solvent such as tetrahydrofuran;
c3/ allowing the amine derivative obtained to react with a nucleophilic selenium derivative, optionally generated in situ, in the presence of a copper Cu(I) salt, in a polar organic solvent, to lead to the corresponding (hetero)arylisoselenazine derivative;
d3/ optionally, N-alkylating or N-arylating or N-acylating or N-sulphonylating, according to conventional procedures, said corresponding (hetero)arylisoselenazine derivative;
finally, optionally, for the preparation of compounds of formula (I) in which n=1, Se-oxidising, according to conventional procedures, the corresponding derivative of formula (I) (wherein n=0) obtained previously according to any one of the synthetic routes above.
Said method is close to that described in the application WO-A-95/27706. In a way, it constitutes an adaptation of said method according to WO-A-95/127706 for obtaining heteroaromatic derivatives. The implementation of each of its steps does not give rise to any particular difficulty to the person skilled in the art.
According to the advantageous variants of implementation of said method:
the nucleophilic selenium derivative (which intervenes in steps d1-1/, c1-2/, c2/ and c3/g) is a selenocyanate salt, such as potassium selenocyanate for example, which can be:
either generated in situ from selenium metal Se(0) and a cyanide salt, such as potassium cyanide for example,
or added to the reaction medium as such;
the copper salt Cu(I) (which intervenes in the same steps) is cuprous iodide;
the polar organic salt (which intervenes in the same steps) is dimethylformamide;
the intervening oxidant for the Se-oxidation is either peracid, such as meta-chloroperbenzoic acid or hydrogen peroxide.
Other aims, characteristics and advantages of the invention will appear clearly in the light of the following explanatory description made with reference to various non-limiting examples given solely as illustration and which in no way limit the scope of the invention. In the examples, all percentages are given by weight unless otherwise indicated.